absent, small, or homeotic discs 2


Activation and repression activities of ash2 in Drosophila wing imaginal discs

Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. The patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. These results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development (Angulo, 2004).

Loss of ash2 function causes differentiation of ectopic vein tissue, indicating that ash2 is required for intervein development, where it functions as an activator of the intervein-promoting genes net and bs, restricting rho expression to vein regions. In addition, the loss-of-function phenotypes of Egfr alleles are rescued in ash2 mutants, while the gain-of-function phenotypes are enhanced. Furthermore, rho mRNA exhibits an expanded expression pattern in ash2 mutant tissues. Thus, ash2 promotes the maintenance of intervein fate, either by activation of net and bs or by repression of the Egfr pathway. Since rho and bs/net expression is mutually exclusive, it cannot be determined whether the Ash2 complex interacts directly with one or all of them. However, since bs expression is inhibited by the loss-of-function of ash2 during larval and pupal stages, it can be proposed that ash2 acts as a long-term chromatin imprint of bs that is stable throughout development (Angulo, 2004).

The results in adult clones and from analysis of genetic interactions suggest that ash2 acts principally by maintaining B and D intervein regions, since the C intervein remains unaltered in ash2 mutants. This region is under the control of organising genes that respond to the Hh signal. One of these genes is kn, which prevents vein differentiation in the C intervein and is required for the expression of bs in this domain. bs expression is regulated by two enhancer elements: the boundary enhancer, which is dependent on hh and controls bs expression in the C intervein region through kn; and another enhancer dependent on Dpp activity, which controls bs expression in B and D intervein domains. Thus, the role of ash2 as a positive regulator of bs is mainly restricted to regions beyond the kn domain where the Dpp dependent bs enhancer is active (Angulo, 2004).

It has been found that some combinations of dpp alleles and mosaic clones of sal-C (spalt-major/spalt-related complex of zinc-finger transcription factors) result in elimination of B and D intervein regions, along with fusion of their flanking veins. Although the genetic interactions between ash2 and either bs or net could be the result of a synergistic failure to activate genes downstream of Dpp, the results indicate that this may not be the case because salm is expressed in the central domain of the wing pouch of those mutant combinations (Angulo, 2004).

It has been recently been shown that another trxG complex, the Brm complex, is involved in regulating wing vein development. Components of that complex interact genetically with net and bs at pupal stages to regulate the expression of rho, and the complex is specifically required in cells within and bordering L5 to mediate proper signalling. There are some key differences between the Brm complex and Ash2: (1) Ash2 maintains bs expression from the third instar stage; (2) the Ash2 complex is mainly required for interveins B and D; and (3) the enhancement or suppression phenotypes of the genetic interactions with Egfr and intervein-promoting alleles are much stronger for ash2 than for the Brm complex. Taken together, these results suggest that ash2 plays a crucial role in intervein identity and that each trxG complex acts in a specific spatiotemporal program to maintain organ identity (Angulo, 2004).

The positioning of vein tissues depends on the sal-C patterning dictated by the Dpp signalling pathway. Low levels of sal-C in the anterior compartment are required for the expression of kni-C, which triggers the differentiation of L2. Lack of ash2 activity results in downregulation of salm and upregulation of kni. Thus, it is possible that within the sal-C domain, the ectopic expression of kni is a result of low levels of salm. However, when high levels of salm or salr are maintained by ectopic activation, lack of ash2 nevertheless results in de-repression of kni. Moreover, kni is also cell-autonomously de-repressed by loss-of-function of ash2 in cells outside of the sal-C expression domain. Thus, the repression state in the whole wing must be maintained by factors other than sal-C. The kni/knirl L2-enhancer is subdivided into activation binding sites for Brk, En and the Sd/Vg complex, and repression binding sites for Sd/Vg, En, Salr and Brk. No changes were observed either in ß-gal expression from the EX-lacZ enhancer or in sd, vg, brk or en expression in clones lacking ash2. Therefore the de-repression of kni in ash2 mutant cells must be accounted for by a mechanism entirely different from that of the signal-dependent induction of L2, perhaps through another enhancer more global than that of L2 (Angulo, 2004).

The low levels of salm expression associated with ash2I1 clones may also be explained by de-repression of kni. In dorsal tracheal cells, kni/knrl activity represses salm transcription, and this repression is essential for branch formation. Similarly the establishment of the border between cells acquiring dorsal branch and dorsal trunk identity entails a direct interaction of Knirps with a salm cis-regulatory element. Also in the wing, kni and knrl are likely to refine the L2 position by positive auto-regulation of their own expression and by providing negative feedback to repress salm expression (Angulo, 2004).

It is possible that the de-repression of kni, intervein inhibition and appearance of extra vein tissues are linked events. The kni-C complex organises the development of the L2 vein by activating rho and inhibiting bs. Thus, kni-C participates in L2 morphogenesis by functioning downstream of salm and upstream of vein-intervein genes. The ectopic activation of kni by lack of ash2 could trigger intervein repression and vein activation. Indeed, ectopic activation of UAS-kni results in broad expression of rho and elimination of Bs expression in pupal wings, leading to the production of solid vein material. However, in adult clones not all ash2 mutant cells develop vein tissue. This raises the possibility that de-repressed kni may not be fully functional, since ectopic kni is often localised to the cytoplasm rather than the nucleus. Alternatively, ash2 could have independent functions in the wing, maintaining the repressed state of kni alongside maintenance of the intervein condition, by acting on different targets (Angulo, 2004).

The ash2112411 mutation can partially rescue the loss of L2 in kniri-1 mutants. This is in contrast to the observation that the L2 enhancer appears not to mediate the effect of ash2. The kniri-1 allele is a 252 bp deletion in the enhancer of L2. It has been shown, however, that it is possible to rescue the vein-loss phenotype of kniri-1 by expressing a UAS-rho transgene in L2. In addition, double mutant flies for kniri-1 and net partially rescue L2. It is therefore likely that the antagonistic effect of ash2 on rho could account for the partial rescue of L2 in kniri-1 ash2112411 wings, since rho mRNA is expressed in the rescued L2 (Angulo, 2004).

Some PcG genes are known to be required for the maintenance of kni expression domains in the embryo. It is also likely that some trxG genes or other complexes of trxG proteins, such as the Ash2 complex, may interact with repressor sequences necessary to keep kni expression in an off state beyond L2. Moreover, in a genome wide prediction screen it has been shown that kni contains PRE/TREs. Thus, it is proposed that ash2 acts as regulator of kni expression in the wing through an epigenetic mechanism of cellular memory similar to the trx-G regulation of homeotic genes, albeit that it remains to be seen whether kni is a direct or indirect target of ash2 (Angulo, 2004).

A well-studied mechanism through which to induce and preserve cell identities in wing imaginal discs is the response to gradients of the morphogen Dpp. This raises questions about the extent to which the response to Dpp occurs through concentration-dependent mechanisms or cellular memory. There is compelling evidence in favour of the existence of Dpp gradients that organise the pattern and growth of the wing imaginal disc. Dpp signalling causes a graded transcriptional regulation of brk by an interaction between the Dpp transducers and a brk morphogen-regulated silencer. Thus, brk appears to respond to direct morphogenetic signalling rather than remembering the inputs of previous developmental events. However, whereas activation of salm requires continuous signalling through the Dpp pathway, other targets of Dpp, such as omb, remember exposure to the signal. Stable regulation of other genes involved in wing development, such as kni repression, and net and bs activation, would also respond to the cellular memory conferred by epigenetic marks of the Ash2 complex. Thus, both mechanisms -- morphogen-dependant, which will be required for growth and patterning, and epigenetic, which will keep specific genes in an off or on state -- are likely to act simultaneously to maintain cellular identities within the wing (Angulo, 2004).

Because many developmental regulators are only expressed transiently during development, the function of epigenetic complexes is likely to be very dynamic. The developmental events required for the construction of the wing, as with many other morphogenetic events, cannot only rely on an on or off state of gene expression. Instead, morphogenesis is rather malleable and epigenetic marks could act as a means to facilitate, rather that fix, the preservation of developmental fates. It may well be that the epigenetic marks of the Ash2 complex allow changes in chromatin structure to assist the access of proteins that activate or repress gene expression. From an epigenetic point of view, the ultimate refinement of morphogenesis and maintenance of cellular memory will depend upon the interaction of these chromatin remodelling complexes with the factors that trigger or inhibit transcription (Angulo, 2004).

Transcriptional network controlled by the trithorax-group gene ash2

In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).

In the work presented in this study, the allele ash2I1, obtained after excision of the P-lacW transposon present in line l(3)12411, was used. It is lethal in early pupa, and homozygous larvae have reduced and abnormal imaginal discs and brain. The molecular alterations present in the ash2I1 allele are small changes (2-bp deletion and 5-bp insertion) in the fourth intron of the gene. Northern blot analysis of poly(A) RNA extracted from third instar larvae showed the presence of two transcripts (2 and 1.4 kb) with potential coding sequence in WT and only the small one in ash2I1 mutant flies. The longer transcript would account for the already described Ash2 protein, and 5'-rapid amplification of cDNA ends; results supported by in silico predictions from GENEID and GENSCAN show that the 1.4-kb transcript, identical in both WT and ash2I1, contains exons 5-8 present in the previously described ash2 transcript plus a novel 62-bp exon containing 28-bp encoding for amino acids. If translated, the resulting protein would be 350 aa in length and would lack the proline-, glutamic acid-, serine-, and threonine-rich region sequence and the putative double zinc-finger domain, also found in other trx-G proteins. Developmental Northern blot of WT flies showed that both transcripts are present at all stages of development except in early embryos, where only the long maternal transcript was detected. Because the insertion (TTAGG) detected in the fourth intron of the ash2I1 allele creates a putative splicing acceptor site that could generate a transcript containing a premature translation termination codon, it is tempting to speculate on a nonsense mediated decay of such RNA species. To confirm that the mutant behaves as a true trx-G mutant, genetic mosaics were generated in haltere and leg imaginal discs with the aid of the flipase-flipase recombination target (FLP-FRT) technique and the expression of Ultrabithorax (Ubx) was examined by immunohistochemistry. The down-regulation of Ubx accumulation in the homozygous ash2I1 tissue proves this mutant behaves as a trithorax mutant regarding homeotic function (Beltran, 2003).

To identify downstream genes of ash2 function, the population of mRNA species isolated from homozygous ash2I1 third instar larvae was compared with that of stage-matched WT. Four completely independent cDNA microarray experiments were carried out with poly(A) RNA isolated from separate extractions. The microarrays were constructed by using the ESTs from the Drosophila gene collection 1.0, which contains about one-third of the Drosophila genes. This collection lacks some genes known to be regulated by ash2 such as Ubx. A total of 5,139 cDNAs with a different FlyBase identifier were printed; 4,163 of them passed the quality filters in at least two of the experiments and could be used in the analysis. With a false discovery rate of ~0.025 and a fold change threshold of 1.75, 235 genes were identified of the 4,163 (5.6%) whose expression levels change significantly in the mutant, pointing to ash2 as a putative regulator of them. One hundred forty of these genes were positively regulated and 95 negatively regulated. Down-regulated genes include ash2 with a 1.76-fold change, a rather high value if the presence of the 1.4-kb transcript in the ash2I1 mutant is acknowledged. The differential expression levels of candidate genes was examined by performing semiquantitative RT-PCR analysis on selected genes (Beltran, 2003).

Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Focus was placed on the up-regulated gene FasII, and clonal analysis was performed on a Minute background, to assess whether the behavior of the FasII transcript observed with this ash2 mutant was also kept at the protein level in wing imaginal discs. Homozygous mutant cells show a clear up-regulation of FASII, mainly in the wing pouch area further away from the dorsoventral margin, where FASII was found to be very slightly expressed in WT wing discs. The up-regulation of FasII and other cell adhesion molecules like mfas, together with the up-regulation of the transcription factor vri, could explain some of the phenotypes previously found by clonal analysis, such as disruption of vein-intervein patterning, because it is known that preferential accumulation of specific adhesion molecules characterizes the final stages of vein differentiation. Furthermore, because FASII is involved in the development of the neural system, its pattern of expression in ash2I1 mutant brains was compared with that of WT; they present a distorted phenotype in the optic lobes as well as fasciculation defects in the ventral ganglion, a process in which FASII plays a central role. A gain-of-function screen (Kraut, 2001) identified ash2 and FasII as genes involved in the development of the neural system, further supporting a relationship between them (Beltran, 2003).

In a search for putative downstream genes for Set1, many genes involved in transcriptional regulation of growth and cell cycle control have been found (Nislow, 1997). According to the current results, ash2 also seems to act on genes that fall into these classes. For example, Eip75B, vri, jing, or HmgD could be classified in the first class (transcriptional regulation), whereas CyclinA (CycA), cutlet, Pu, mei-S332, or mus209 could do so in the second (cell cycle control). It was confirmed, by clonal analysis in wing imaginal discs, that the protein product of the down-regulated gene CycA is present to a lesser extent in mutant clones. The down-regulation of CycA can play a role in the proliferation defects observed in mutant cells when clones are generated in the imaginal discs. Furthermore, in genetic mosaics, homozygous mutant ash2I1 cells show effects on both cell differentiation and cell size. Normal wing cells develop a single hair, whereas ash2I1 cells can develop either a single or multiple hairs. In addition, the spacing between ash2I1 mutant cells is increased compared with that of WT ones, suggesting that ash2I1 mutant cells are larger (Beltran, 2003).

Preliminary promoter analysis of the regulated genes does not seem to reveal any defined and clear pattern. Although this needs further investigation, it may be difficult to find any consensus sequence, mainly because the regulated genes reflect all of the transcripts present at that moment and therefore a particular cellular status. Even though some authors have described cis-acting Trithorax Response Element sequences needed for some trx-G proteins to exert their function, it is not yet possible to discern between primary and secondary targets of Ash2 on the basis of this information. However, it can be stated that Ash2 function is required for a wide variety of biological processes during larval development (Beltran, 2003).

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. This study identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). The results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. The occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription was characterized. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, specific motifs were identified associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. The data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control (P´rez-Lluch, 2011).

Work using various model organisms and cultured cells has provided high-resolution profiles of histone modifications and transcription factor binding across different genomes. This study used direct sequencing of ChIP DNA from wing disc to analyse ASH2 function. Because the cell composition of isolated wing disc tissue is rather homogeneous, it has been possible to set apart several attributes. First, ASH2 occupancy correlates with the presence of phosphorylated forms of RNA Polymerase II and activating histone marks in expressed genes. But, a direct role for ASH2 in gene repression as well cannot be dismissed, since ASH2 also targets silenced genes. In support of this, ASH2-interacting proteins HCF-1 and dMyc are involved in both transcriptional activation and repression. Alternatively, silenced ASH2 target genes could be arrested in an intermediate ready-to-go state of transcription, which may be activated by external signals. Second, the results agree with previous observations in Drosophila and Xenopus embryos, where dually marked domains do not seem to be a common feature. It has been reported that bivalently marked chromatin, containing both H3K4 and H3K27 trimethylation, is a hallmark of developmentally regulated silenced promoters in mammalian embryonic stem cells. In contrast, these marks can be coupled to the differential expression pattern of several genes throughout the wing disc, therefore indicating the presence of each individual mark in different cells. A recent report using a similar genome-wide approach in undifferentiated cell-enriched Drosophila testis reveals that differentiation-associated genes are also linked with monovalent modifications. Third, ASH2 binding was used together with activating marks of transcription as a powerful tool to identify previously unannotated genes (P´rez-Lluch, 2011).

The actively transcribed genes in the wing disc are occupied by nucleosomes with histone modifications that are hallmarks of both initiation and elongation. This study has uncovered a positive correlation between activating marks of transcription (both H3K4me3 and H3K36me3) and ASH2 occupancy. This study has also determined that ASH2 contributes to H3K4me3 in nearby nucleosomes. H3K4me3 is associated with the TSS of active genes, whereas H3K27me3 spreads over large regions of chromatin to promote silencing and H3K36me3 is found in actively transcribed regions. Only genes containing H3K36me3 undergo further elongation and produce mature transcripts (P´rez-Lluch, 2011).

Transcriptional regulation is a multistep process controlled by a large complex machinery at the level of recruitment, initiation, pausing and elongation of RNA Polymerase II. A series of recent genome-wide studies indicate that many developmental and inducible genes, prior to their expression, contain RNA Polymerase II bound predominantly in their promoter proximal regions in a stalled state. Nevertheless, not only silenced genes show an enrichment of the RNA Polymerase II density at their TSS as the stalled polymerase is also present at this region in active genes. The presence of ASH2 and H3K4me3 together with PolIIS5P at the TSS of expressed genes is consistent with previous reports proposing that promoter-proximal stalling serves not only to fully repress but also to attenuate transcription of active genes. As recently described, transient stalling of polymerase is a general feature of early elongation, even in highly active genes (P´rez-Lluch, 2011).

The analysis of ash2 mutant flies indicates that ASH2 is performing its canonical function promoting H3K4me3, regardless of the effect on the transcriptional state of its target genes and the context specificity of its recruitment to promoters. In light of the results obtained with RNA Polymerase II modifications in the mutants, it is concluded that ASH2 influences different aspects of transcription. The specific binding motifs identified in differentially regulated genes, together with the co-occupancy of ASH2 and PolIIS5P at the TSS, suggests a role in transcription initiation. Nevertheless, the reduction of PolIIS5P in mutant flies points to a fast escape from stalling in the absence of ASH2 (P´rez-Lluch, 2011).

Distinct sets of accessory factors are associated with polymerase stalling and its escape from this state, acting either by direct interaction with RNA Polymerase II, or by manipulating the chromatin environment. Among these factors, there are proteins associated with polymerase stalling, such as the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF), and others that contribute to escape from stalling, such as the positive transcription-elongation factor-b (P-TEFb) complex and the general transcription factors TFIIS and TFIIF. It remains to be elucidated whether ASH2 interacts directly with some of these factors. However, NELF and GAF have been found linked to promoter-proximal pausing at many genes in Drosophila. A connection between ASH2 and polymerase stalling in developmental genes could, therefore, be envisioned through GAF, since it is known that GAF is a recruiter of PcG and trxG complexes to DNA. In fact, about half of the downregulated genes in ash2 mutants presenting GAGA sites are NELF targets. Furthermore, it has been recently reported that c-Myc regulates RNA Polymerase II pause release by recruiting P-TEFb to its target genes, and it is known that ASH2 interacts with Myc in flies. The enrichment of Ebox and Mnt/Max motifs found in upregulated genes in ash2 mutants (see Characterization of ASH2 binding regions) points to a function of ASH2 through Myc in their transcriptional regulation. A subset of these motifs was characterized in H3K4me3 regions. It has been possible to associate these motifs with downregulated and upregulated genes in ash2 mutants, suggesting differential transcriptional regulation (P´rez-Lluch, 2011).

Several effector proteins that can bind to H3K4me3 determine the functional outcome of this histone modification. The activities of these binding proteins range from activation and repression of transcription, chromatin remodelling or splicing efficiency among others. An additional role for ASH2 during transcript elongation and maturation should not be excluded. Indeed, it has been suggested that methylated H3K4 serves to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components. The fact that downregulated and upregulated genes in ash2 mutants display clear differences in size and genomic organization (gene size, alternative isoforms and number of exons) suggests they may be regulated in a different way during transcription and processing of RNA. Finally, recent reports indicate an association of RNA Polymerases II and III at promoter regions of housekeeping genes and a recruitment of RNA Polymerase III through Myc interacting with the cofactor BRF has also been described. However, preliminary experiments discard the implication of other polymerases in the transcription of these housekeeping genes in the absence of ASH2. Taken together, these results support a model in which an ASH2-containing complex would act at different levels of transcriptional regulation (P´rez-Lluch, 2011).

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3 was analyzed. ChIP-seq (Little imaginal discs) results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling shows that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5) form. The results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription (Lloret-Llinares, 2012).

This study reports that dKDM5/LID localizes at TSS of developmental genes and regulates H3K4me3. dKDM5/LID target genes are actively transcribed and, though weakly, they are significantly downregulated in lidRNAi mutant flies. Previous reports already suggested a positive contribution of dKDM5/LID to transcriptio. The current results also show that dKDM5/LID target genes are bound by ASH2, an evolutionarily conserved component of H3K4-KMT2 complexes that localizes at TSS and is required for H3K4me3. In addition, dKDM5/LID target genes are strongly downregulated in ash2 mutant flies. These observations indicate that dKDM5/LID and ASH2 act coordinately to regulate H3K4me3 at TSS of developmental genes for their efficient transcription. dKDM5/LID and ASH2, however, do not bind chromatin simultaneously, indicating that they act at different moments during transcription. These observations strongly favor a model by which ASH2 and dKDM5/LID act sequentially during transcription to facilitate its progression. On this regard, work performed in budding yeast links chromatin modification events to sequential RNApol II activation. At a first step, TFIIH-mediated phosphorylation of CTDSer5 recruits scKMT2/SET1 to methylate H3K4, and induces promoter escape. Later, the onset of productive transcription involves phosphorylation of CTDSer2, which results in recruitment of H3K36 KMT3/SET2 both in budding yeast and mammals. dKDM5/LID recruitment might also be regulated during transcription cycle progression. In this context, it is possible that, after RNApol II activation and subsequent H3K4-methylation, dKDM5/LID is recruited and transient demethylation resets chromatin to the original 'unmethylated' state, facilitating the next RNApol II molecule to initiate progression through the transcription cycle. Consistent with this model, it was shown that the C-terminal PHD-finger of dKDM5/LID, or the mammalian homolog KDM5A/JARID1A, specifically binds H3K4me2,3 (Wang, 2009) and, furthermore, this study has shown that dKDM5/LID binds chromatin enriched in H3K4me3, whereas chromatin bound by ASH2 is poor in H3K4me3. Finally, the results also show that dKDM5/LID depletion significantly reduces RNApol ll occupancy, in particular by the promoter-proximal Pol IIoser5 active form, providing a basis for the positive contribution of dKDM5/LID to transcription. In contrast, occupancy by the elongating Pol IIoser2 form is not similarly affected, showing a tendency to be slightly increased. It is possible that, in the absence of dKDM5/LID, constitutive/increased H3K4me3 at TSS affects RNApol II pausing and, hence, transcription efficiency. Actually, it has been shown that depletion of NELF, a factor required for RNApol II pausing, results in a general downregulation of its target genes both in Drosophila and human cells (Lloret-Llinares, 2012).

Several reasons could account for the weakness of the observed effect of dKDM5/LID depletion on gene expression. On one hand, though dKDM5/LID content is strongly reduced in lidRNAi, depletion is not complete. Note that null lid mutations could not be used, as they are lethal during late embryo/early larvae development. Second, although dKDM5/LID is the only enzyme known to specifically demethylate H3K4me3 in Drosophila, additional KDMs might exist capable of playing a similar function. At this respect, it was reported that dKDM2, which was originally found to demethylate H3K36me2, might also be capable of demethylating H3K4me3. Thus, it is possible that loss of dKDM5/LID is partially compensated by dKDM2. As a matter of fact, a genetic interaction was recently reported between dKDM5/lid and dKDM2 (Lloret-Llinares, 2012).

The proposed function of dKDM5/LID in the regulation of transcription is likely conserved, as it was recently reported that mammalian KDM5B/JARID1B preferentially localizes at TSS of developmental genes and regulates H3K4me3. Mammalian KDM5C/JARID1C has also been shown to bind at TSS. Interestingly, although KDM5B/JARID1B is required to efficiently silence stem and germ cell specific genes during neuronal differentiation, its depletion in ESCs shows also a weak downregulation of target genes. In Drosophila, dKDM5/LID has also been shown to be involved in repression of some developmental genes. In fact, in the wing imaginal disc, ∼20% of dKDM5/LID target genes show no detectable H3K4me3. Altogether, these observations suggest that dKDM5/LID might play a dual function; repressing specific genes during development and, in differentiated cells, regulating H3K4me3 dynamics at TSS during transcription (Lloret-Llinares, 2012).

Protein Interactions

Characterization of skittles, an Ash2 interactor

The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. A Drosophila PIP5K type I (skittles) has been cloned and mutations in this gene have been generated. This analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, no evidence was found for PIP5KI involvement in neural secretion (Hassan, 1998).

The Drosophila trithorax group proteins BRM, Ash1 and Ash2 are subunits of distinct protein complexes

To determine if Brm physically interacts with other trithorax group proteins, the Brm complex was purified from Drosophila embryos and its subunit composition analyzed. The Brm complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the Brm complex are not encoded by trithorax group genes. The proteins that consistently copurify with Brm have been designated Brm-associated proteins (BAPs) and are referred to by their molecular mass in kDa (BAP45, BAP47, BAP55, BAP60, BAP74, BAP111 and BAP155). Two different purification schemes identify the same set of seven polypeptides associated with Brm (Papoulas, 1998).

Biochemical evidence is presented for the existence of two additional complexes containing trithorax group proteins: a 2 MDa Ash1 complex and a 500 kDa Ash2 complex. Based on their genetic properties, three of the best candidates for trx-G members that physically interact with Brm are Absent, small or homeotic discs 1 and 2 (Ash1 and Ash2), and Trithorax. In spite of being bona fide members of the trx-G, neither Ash1, Ash2 nor Trithorax are found to be a part of the Brm complex. Affinity-purified polyclonal antibodies against Ash1 detect three prominent bands in embryo extracts, the largest of which is 270 kDa. The predicted size of the Ash1 protein (244 kDa) and the variability in amount of the smaller bands detected in different experiments argues that the 270 kDa band represents full-length Ash1 and that the smaller bands are degradation products. Affinity-purified antibodies against ASH2 detect a single band of 94 kDa. Although the Brm, BAP45/Snr1, Ash1 and Ash2 proteins are readily detected by western blotting in whole embryo extracts, neither the Ash1 nor Ash2 proteins are detected in purified Brm complex. Similar experiments using antibodies against Trx did not yield reproducible results, presumably due to the low abundance and instability of this >350 kDa protein. An examination to see if Ash1 or Ash2 are physically associated with Brm in embryo extracts used a coimmunoprecipitation assay. Neither Ash1 nor Ash2 were found to coimmunoprecipitate with Brn. It is therefore concluded that the Ash1 and Ash2 proteins do not stably interact with the Brm complex. To determine whether Ash1 and Ash2 are components of protein complexes distinct from the Brm complex in the Drosophila embryo, the native molecular mass of both proteins was examined by gel filtration chromatography. The ASH1 protein has a native molecular mass of approximately 2 MDa. By contrast, Ash2 has an apparent native molecular mass of approximately 500 kDa. No monomeric Ash1 or Ash2 is detected in embryo extracts. It is concluded that the Drosophila embryo contains at least three distinct protein complexes containing trx-G proteins: the 2 MDa BRM complex, a 2 MDa Ash1 complex and a 500 kDa Ash2 complex (Papoulas, 1998).

The COMPASS family of H3K4 methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila Trithorax (Trx). While humans have six COMPASS family members, Drosophila has a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). This study reports the biochemical purification and molecular characterization of the Drosophila COMPASS family. A one-to-one similarity occurs in subunit composition with their mammalian counterparts, with the exception of (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. This study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila (Mohan, 2011).

Histone H3 lysine 4 methylation (H3K4me) is associated with the transcriptionally active regions of the genome in yeast, flies, and mammals. Set1 was identified as a component of a macromolecular protein complex named COMPASS (complex of proteins associated with Set 1), as the first H3K4 methylase, and it is responsible for all mono-, di-, and trimethylation of H3K4 in yeast. In Drosophila, four SET domain-containing proteins, namely, Trithorax (Trx), Trithorax-related (Trr), dSet1, and Ash1, have been reported to implement H3K4 methylation. All but Ash1, which has subsequently been demonstrated to be an H3K36 methyltransferase, are related to subunits of the six COMPASS and COMPASS-like complexes in mammals. trx was originally characterized as a gene that when mutated caused homeotic transformations. Detailed genetic and molecular analyses showed that Trx is required to maintain activation states of its target genes throughout development and counteracts the repressive effects of the Polycomb group proteins (PcG). Trr was identified based on sequence similarity to Trx but was shown to function in the regulation of hormone-responsive gene expression (Sedkov, 2003). dSet1 was identified based on sequence homology to the Saccharomyces cerevisiae and mammalian Set1 proteins (Mohan, 2011).

In mammals, there are at least six SET1-related proteins that form COMPASS-like complexes, namely, SET1A, SET1B, and MLL1 to MLL4. SET1A and SET1B are orthologous to dSet1; MLL1 and MLL2 are orthologous to Drosophila Trx; MLL3 and MLL4 (also known as ALR) are orthologous to Drosophila Trr (Mohan, 2010; Shilatifard, 2008; Smith, 2010). All of the mammalian COMPASS family of H3K4 methylases share ASH2L, RBBP5, DPY30, and WDR5 as common components. Analysis of the mammalian complexes allows classification into three classes based on unique components within each class: COMPASS, represented by SET1A and SET1B, contains WDR82 and CXXC1, proteins implicated in regulating trimethylation by yeast COMPASS; the MLL1/2 complexes contain Menin, implicated in targeting MLL1 to the Hox genes; the MLL3/4 complexes contain PTIP, PA-1, and NCOA6 (Cho, 2007), which are important for the gene-specific targeting of these complexes, and UTX, a histone H3K27 demethylase thought to be involved in counteracting PcG-mediated gene silencing (Eissenberg, 2010: Hughes, 2005; Lee, 2007; Mohan, 2011 and references therein).

This study purified and characterized the dSet1, Trx, and Trr complexes. In contrast to a previous report that Trx formed a heterotrimeric complex with CBP and SBF1, this study found instead that Trx forms a COMPASS-like complex containing orthologs of all known components of the MLL1 complex in mammals. These studies also demonstrate that Drosophila Set1 is the major contributor to the bulk in vivo dimethylation and trimethylation of H3K4 and that this depends on a conserved form of histone cross talk, where monoubiquitinated H2B is required for H3K4 trimethylation by dSet1. It was also found that mammalian MLL3/4 are represented in flies by two genes, Trr and LPT, and that the encoded proteins exist together in a COMPASS-like Trr complex. Taken together, this evidence for the existence of one representative complex in Drosophila for each of the three classes of the six COMPASS family proteins in mammals provides a unique opportunity to discover the differences in the targeting and function of H3K4 methylation by these complexes (Mohan, 2011).

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). This study reports the biochemical purification and molecular characterization of the Drosophila COMPASS family. A one-to-one similarity in subunit composition with their mammalian counterparts was observed, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. This study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila (Mohan, 2011).

Modifications of histones and the protein machinery for the generation and removal of such modifications are highly conserved and are associated with processes such as transcription, replication, recombination, repair, and RNA processing. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites in all eukaryotes tested and is generally believed to be a hallmark of active transcription. The H3K4 methylation machinery was first identified in yeast and named Set1/COMPASS. Six H3K4 methyltransferase complexes have been identified in humans, including SET1A/B, which are subunits of human COMPASS, and MLL1 to MLL4, which are found in COMPASS-like complexes (Mohan, 2011).

Although Trx and Trr were identified quite some time ago, their relative contributions to different states of overall H3K4 methylation were not known. Studies of human cells and Drosophila cells has shown that SET1 is the major contributor of H3K4 trimethylation levels in cell. During the preparation of the manuscript, a study of Drosophila also showed that dSet1, as a part of COMPASS, is responsible for the majority of H3K4 di- and trimethylation (Ardehali, 2011), which is in line with the findings presented in this study. These findings suggest that dSet1 could be responsible for the deposition of H3K4 trimethylation at the transcription start sites of the most actively transcribed genes as a consequence of postinitiation recruitment via the PAF complex (Smith, 2010: see Recruitment of histone-modifying activities by RNA Pol II). Trx and Trr both show extensive distribution along polytene chromosomes, although neither protein is required for bulk levels of H3K4me3. Perhaps Trx and Trr implement H3K4 methylation in a more gene-specific manner, at distinct stages of transcriptional regulation, or alternatively, have other substrates or functions (Mohan, 2011).

These biochemical studies have demonstrated that the Drosophila complexes are very similar to their mammalian counterparts in subunit composition. These studies have also demonstrated the utility of a baculovirus superinfection system for expressing proteins in Drosophila cells. Large-scale transient transfections offer several potential advantages over generating clonal stable cell lines, one of which is that the overexpression of some proteins could be toxic to cells. This can be a problem even when using inducible promoters, such as the Mtn promoter, due to leaky expression under uninduced conditions. Moreover, the baculovirus infection and expression strategy took about 3 weeks from the cloning of the cDNA into the viral vector, generating the virus, infection of S2 cells, and purification of the complexes from nuclear extracts. In contrast, conventional cloning took 4 months from cloning the cDNA into the vector to generating and characterizing the clonal cell lines. FLAG-HA-dWDR82 was purified from both stably transfected S2 cells and from the superinfection system and both strategies yielded a strikingly similar enrichment of target proteins (Mohan, 2011).

All of the COMPASS family members in Drosophila have several common subunits, namely, Ash2, Rbbp5, Wdr5, and Dpy30, which are homologs of CPS60, CPS50, CPS30, and CPS25, respectively, as well as each having complex-specific subunits. Many of these subunits have established, conserved roles in both the yeast and mammalian complexes: ASH2L is required for proper H3K4 trimethylation, as is CPS60 in yeast; both WDR5 in humans and CPS30 in yeast are required for the mono-, di-, and trimethylation of H3K4, and each is required for proper formation of the COMPASS and MLL complexes. Conservation of this degree in the H3K4 methylation machinery suggests that Drosophila might have similar machinery. However, it had previously been reported that Trx forms a complex with CBP and SBF, but no corresponding complexes have been found in mammals (Mohan, 2011).

The demonstration of the presence of shared components between COMPASS and COMPASS-like complexes in Drosophila supports the findings that these proteins are required for the proper functional architecture critical for the methylation of H3K4. The complex-specific components found in association with the dSet1, Trx, and Trr complexes further demonstrate a one-to-one correspondence of subunits between the Drosophila and human COMPASS family members that will allow the use of Drosophila as a model system for understanding the function of the human complexes. For example, while Set1/COMPASS is conserved from yeast to humans, it is possible that the metazoan complexes have additional functions needed for development. As the subunit compositions of both the SET1A and SET1B complexes are identical, it is likely that their functional analysis would be hindered by redundancy between the two complexes. The presence of a single dSet1 complex in flies may serve as an excellent starting point to dissect the metazoan-specific functions of the SET1 complexes (Mohan, 2011).

MLL-related proteins are multidomain proteins with the capacity to bind to many other proteins that may modulate their function. For example, Menin binds to the extreme N terminus of MLL1/2 and is required for proper targeting of the MLL1/2 complex to chromatin. Owing to its conserved components and interactions, but nonredundant nature, investigation of the Drosophila Trx complex promises to aid in our understanding of the MLL1 and MLL2 complexes, specifically in their role in development (Mohan, 2011).

Currently there is very limited understanding of the functions of the various domains within the MLL3/4 proteins. The identification of LPT, which is homologous to the N terminus of MLL3/4, as a component of the Trr complex indicates the importance of PHD fingers residing in the LPT protein for the proper functioning and/or targeting of the Trr complex to chromatin. This separation of the MLL3/4 protein in Drosophila as Trr and LPT could allow dissection of the functions of N and C termini. Various studies have identified mutations in MLL3, MLL4, and UTX in a variety of cancers. Therefore, studies of the LPT-Trr complex could improve understanding of the targeting and regulation of these complexes with relevance to human disease (Mohan, 2011).

Importantly, Drosophila has a single representative of each class of COMPASS family members found in mammals, in which two representatives of each complex exist. In contrast, nematodes, such as the genetically tractable C. elegans, contain only a Set1 and MLL3/4-related protein, but no MLL1/2 representative. Given the power of genetic manipulation, the identification of the COMPASS, Trx, and Trr complexes in Drosophila that share similar subunits with their mammalian counterparts will greatly facilitate an understanding of the biological functions of the H3K4 methylation machinery in development and differentiation (Mohan, 2011).

absent, small, or homeotic discs 2: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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